This proposal is focused on the creation and preclinical evaluation of a novel vaccine containing a recombinant, chimeric polypeptide antigen (rCPA) combined with the most immunogenic type of glucan particles (GPs) as an adjuvant and delivery platform in a humanized murine model of coccidioidomycosis. Preliminary data have revealed that vaccine immunity to Coccidioides infection is dependent on both MyD88- and Card9-mediated Th17 and Th1 responses. Furthermore, Th17 immunity is indispensable. GPs are hollow, highly purified yeast cell walls composed predominantly of -1,3-glucan that has been shown to stimulate Th17 and Th1 immunity. Preliminary studies demonstrate robust and long-lasting adaptive T-cell responses following immunization of mice with GPs loaded with antigens. The central hypothesis is that a multivalent antigen (rCPA) incorporated into the most immunogenic form of GPs, when combined with a ligand(s) that elicits Toll- like receptor (TLR)- or C-type lectin receptor (CLR)-mediated immune responses will augment vaccine efficacy by stimulating robust and durable Th17 and/or Th1 immunity to Coccidioides infection. The two Specific Aims are: Aim 1. To evaluate immunogenicity of rCPA and to optimize antigen design. Multivalent Coccidioides vaccines containing multiple antigens have been shown to be more effective in stimulation of protective immunity than individual antigens. The proposed rCPA that incorporates 4 protective antigens of Coccidioides has been successfully expressed and purified. The immunogenicity of these individual antigens and rCPA will be evaluated using human MHC II-expressing HLA-DR4 transgenic mice and peripheral blood monocytic cells isolated from skin test-positive and -negative volunteers to a clinically-approved antigen of Coccidioides. The design of the rCPA will be optimized to contain only the immunogenic polypeptides among the 4 selected antigens that can stimulate all arms of vaccine immunity including activation of IL-17A- and IFN-?-producing T cells and antibody production. Aim 2. To identify an effective adjuvant ligand(s) combined with modified GPs to enhance rCPA vaccine immunity to Coccidioides infection. The optimized rCPA will be loaded into 4 different types of glucan/mannan/chitin particles (GxP) and tested individually for protective efficacy in humanized HLA-DR4 transgenic mice. Glucan particles are an experimental vaccine adjuvant under clinical trials. The most protective type of GPs will be co-loaded with a selected ligand(s) for pattern recognition receptors to augment T-cell immunity and protective efficacy. The vaccine(s) that confer resistance to Coccidioides infection will be analyzed to dissect correlates of protection including cytokine expression by T cells and antigen-presenting cells during the efferent and afferent limbs of immune responses after an intranasal challenge with a potentially lethal dose of Coccidioides spores. Upon completion of this project, a novel lead multivalent vaccine candidate will be generated for advancement into clinical trials for assessment of its safety and protective efficacy against coccidioidomycosis.